Virginia A. Zakian

Position Effect at S. cerevisiae Telomeres: Reversible Repression of Pol II Transcription

S. cerevisiae chromosomes end with the telomeric repeat (TG1-3)n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2- cells) containing white sectors (characteristic of ADE2+ cells). Repression was due to proximity to the telomere itself since an 81 bp tract of (TG1-3)n positioned downstream of URA3 when URA3 was approximately 20 kb from the end of chromosome VII did not alter expression of the gene. However, this internal tract of (TG1-3)n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed. These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control.

Telomeres: Beginning to Understand the End

Telomeres are the protein-DNA structures at the ends of eukaryotic chromosomes. In yeast, and probably most other eukaryotes, telomeres are essential. They allow the cell to distinguish intact from broken chromosomes, protect chromosomes from degradation, and are substrates for novel replication mechanisms. Telomeres are usually replicated by telomerase, a telomere-specific reverse transcriptase, although telomerase-independent mechanisms of telomere maintenance exist. Telomere replication is both cell cycle- and developmentally regulated, and its control is likely to be complex. Because telomere loss causes the kinds of chromosomal changes associated with cancer and aging, an understanding of telomere biology has medical relevance.

Loss of a yeast telomere: Arrest, recovery, and chromosome loss

Yeast strains were constructed in which a single telomere could be eliminated from the end of a dispensable chromosome. In wild-type cells, elimination of a telomere caused a RAD9-mediated cell cycle arrest, indicating that telomeres help cells to distinguish intact chromosomes from damaged DNA. However, many cells recovered from the arrest without repairing the damaged chromosome, replicating and segregating it for as many as ten cell divisions prior to its eventual loss. Telomere elimination caused a dramatic increase in loss of the chromosome in all strains examined, demonstrating that yeast telomeres are also essential for maintaining chromosome stability. Thus, in spite of checkpoint and DNA damage repair systems, many chromosomes that lose a telomere are themselves destined for loss.

DNA secondary structures: stability and function of G-quadruplex structures

In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.

Telomere-Telomere Recombination Is an Efficient Bypass Pathway for Telomere Maintenance in Saccharomyces cerevisiae

ABSTRACT Many Saccharomyces telomeres bear one or more copies of the repetitive Y′ element followed by ∼350 bp of telomerase-generated C1–3A/TG1–3 repeats. Although most cells lacking a gene required for the telomerase pathway die after 50 to 100 cell divisions, survivors arise spontaneously in such cultures. These survivors have one of two distinct patterns of telomeric DNA (V. Lundblad and E. H. Blackburn, Cell 73:347–360, 1993). The more common of the two patterns, seen in type I survivors, is tandem amplification of Y′ followed by very short tracts of C1–3A/TG1–3 DNA. By determining the structure of singly tagged telomeres, chromosomes in type II survivors were shown to end in very long and heterogeneous-length tracts of C1–3A/TG1–3 DNA, with some telomeres having 12 kb or more of C1–3A/TG1–3 repeats. Maintenance of these long telomeres required the continuous presence of Rad52p. Whereas type I survivors often converted to the type II structure of telomeric DNA, the type II pattern was maintained for at least 250 cell divisions. However, during outgrowth, the structure of type II telomeres was dynamic, displaying gradual shortening as well as other structural changes that could be explained by continuous gene conversion events with other telomeres. Although most type II survivors had a growth rate similar to that of telomerase-proficient cells, their telomeres slowly returned to wild-type lengths when telomerase was reintroduced. The very long and heterogeneous-length telomeres characteristic of type II survivors in Saccharomyces are reminiscent of the telomeres in immortal human cell lines and tumors that maintain telomeric DNA in the absence of telomerase.

Saccharomyces telomeres acquire single-strand TG1–3 tails late in S phase

Saccharomyces telomeres consist of approximately 300 bp of C1-3A/TG1-3 DNA. Nondenaturing Southern hybridization, capable of detecting approximately 60 to approximately 300 bases of TG1-3 DNA, revealed that yeast telomeres acquired and lost TG1-3 tails, the predicted intermediate in telomere replication, in a cell cycle-dependent manner. TG1-3 tails were also detected on the ends of a linear plasmid isolated from late S phase cells. In addition, a nonlinear form of this plasmid was detected: this structure migrated in two-dimensional agarose gels like a nicked circle of the same size as the linear plasmid, but had considerably more single-stranded character than a conventional nicked circle. The evidence indicates that these circles were formed by telomere-telomere interactions involving the TG1-3 tails. These data provide evidence for a cell cycle-dependent change in telomere structure and demonstrate that TG1-3 tails, generated during replication of a linear plasmid in vivo, are capable of mediating telomere-telomere interactions.

RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability.

The protein encoded by the RAP1 gene of S. cerevisiae binds in vitro to a consensus sequence occurring at a number of sites in the yeast genome, including the repeated sequence C2-3A(CA)1-6 found at yeast telomeres. We present two lines of evidence for the in vivo binding of RAP1 protein at telomeres: first, RAP1 is present in telomeric chromatin and second, alterations in the level of RAP1 protein affect telomere length. The length changes seen with under- and overexpression of RAP1 are consistent with the interpretation that RAP1 binding to telomeres protects them from degradation. Unexpectedly, overproduction of the RAP1 protein was also shown to decrease greatly chromosome stability, suggesting that RAP1 mediates interactions that have a more global effect on chromosome behavior than simply protecting telomeres from degradation. Such interactions may involve telomere associations both with other telomeres and/or with structural elements of the nucleus.

ATM-related genes: what do they tell us about functions of the human gene?

Every time a cell divides, it must duplicate and segregate its chromosomes. Eukaryotic cells carry out these processes with remarkable order and fidelity. For example, in the yeast Saccharomyces cerevisiae, chromosome loss occurs at rates of only-10-5 per cell division. This precision is achieved at four levels. First, chromosomes bear replication origins, centromeres, and telomeres, cis-acting structures essential for their stable maintenance. Second, there are many transacting factors, such as replication enzymes and spindle components, integral to the processes of replication and segregation. Third, there are cell cycle checkpoints, regulatory mechanisms that delay cell cycle progression when defects are detected in essential genetic structures, allowing repair prior to cell division. Finally, there are the repair activities themselves, activities that correct spontaneous or induced damage to the ge-nome. Although checkpoint and repair genes contribute to chromosome stability, genes whose sole function is to detect or repair DNA damage are not expected to be essential in the absence of DNA damage. Some genes act at multiple levels in ensuring genome integrity. For example , in Saccharomyces, DNA polymerase ~ serves not only as an essential replication enzyme but also as an S phase checkpoint gene and possibly as a DNA repair activity (Navas et al., 1995, and references therein). Ataxia telangiectasia (AT) is a human autosomal hereditary disease characterized by a wide spectrum of defects, including cerebellar degeneration, progressive mental retardation , uneven gait (ataxia), dilation of blood vessels (telangiectasia), immune deficiencies, premature aging, and an-100-fold increase in cancer susceptibility (reviewed by Friedberg et al., 1995). Identification of the gene mutated in individuals suffering from AT revealed that most or all cases of this heterogeneous disease are caused by mutation of a single gene called ATM (Savitsky et al., 1995). Since some affected individuals carry ATM alleles that eliminate essentially the entire open reading frame (ORF), ATM is not an essential gene (Savitsky et al., 1995). Although the disease is recessive and relatively rare, heterozygotes, who make up-1% of the population, also have an increased cancer risk. For example, it is estimated that heterozygosity at the ATM locus accounts for >/9% of breast cancers in the United States (Swift et al., 1991; reviewed by Friedberg et al., 1995). Thus, understanding the mechanism of action of ATM is of considerable practical significance. At the cellular level, AT is characterized by chromosome abnormalities, including elevated numbers of spontaneous and radiation-induced chromosome breaks and telo-mere-telomere …

Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process.

Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.

The Saccharomyces Pif1p DNA Helicase and the Highly Related Rrm3p Have Opposite Effects on Replication Fork Progression in Ribosomal DNA

Replication of Saccharomyces ribosomal DNA (rDNA) proceeds bidirectionally from origins in a subset of the approximately 150 tandem repeats, but the leftward-moving fork stops when it encounters the replication fork barrier (RFB). The Pif1p helicase and the highly related Rrm3p were rDNA associated in vivo. Both proteins affected rDNA replication but had opposing effects on fork progression. Pif1p helped maintain the RFB. Rrm3p appears to be the replicative helicase for rDNA as it acted catalytically to promote fork progression throughout the rDNA. Loss of Rrm3p increased rDNA breakage and accumulation of rDNA circles, whereas breakage and circles were less common in pif1 cells. These data support a model in which replication fork pausing causes breakage and recombination in the rDNA.